CH2402 - Imaging Agent Design

8/3/2019 CH2402 - Imaging Agent Design

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CH2402 Ligand Design for Imaging Techniques

Imaging agents are a very important aspect of everyday healthcare, the early and accurate diagnosisof many diseases can have a significant impact on the efficacy of treatments. There are severalimaging modes available (Table 1) but typically those capable of scanning the entire body lack

resolution and those with high resolution are only practical in vitro .

Table 1 - Imaging modalities

Modality Agents Penetration ResolutionComputed Tomography

(CT)None Deep m

Magnetic ResonanceImaging (MRI)

Gd, Dy Deep m

Positron EmissionSpectroscopy (PET)

Radioisotopes Deep mm

Single-Photon EmissionComputed Tomography

(SPECT)

Radioisotopes Deep mm

Fluorescence microscopy Fluorochromes,rhodamines, BODIPYs

Shallow (mm using visiblelight, cm using NIR)

nm

Ultrasound Microbubbles Deep m

Regardless of the modality the issues encountered while developing an imaging agent are oftensimilar;

Toxicity the agent must be biologically safe at the required levels. This is true even of agents used in in vitro diagnoses, the cell functions must not be effected by the agent inorder to provide a clear result

Stability the agent must survive the conditions found in vivo Uptake the agent must be able to access the active site through cell membranes, the gut

wall or the blood-brain barrier as necessary Localisation and distribution the agent must have a high specificity to limit background

noise Circulation/residence time the agent must remain in vivo for long enough to perform a

scan but for as little additional time as possible to prevent any harmful reaction Penetration of signal

The Physical Properties Required for ImagingThere are several common traits in cell morphology worth considering when designing an imagingagent. Cells are wrapped in membranes; these are made of phospholipids and glycolipids and areessentially organic, non-polar structures. To cross these membranes the agent must be eitherlipophilic itself or able to take part in naturally occurring protein transfer processes used to shuttleimportant molecules across. There is also a potential barrier across the cell membrane, cations are,specifically, pumped out of the cell and so the inside of the cell is slightly negatively charged, largecations will become trapped within cells, this has implications on the toxicity and circulation of anagent. Within the cell there are further membranes surrounding organelles, one organelle of


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particular interest is the nucleus, where DNA is found, others include mitochondria and endoplasmicreticulum. The cell itself is full of cytoplasm, this is a water gel containing some salts andmacromolecules (overall solute levels are ~ 500 g L -1), any agent must therefore be built to exploitand survive this aqueous environment.

Fluorescence Imaging of Biological Systems Advantages;

High sensitivity for a relatively low cost, 4000 cf. 5mil for PET imaging Able to achieve specific staining with multi-channel detection Fluorescence agents provide information on the environment, they are responsive, they

can provide information on conditions such as pH or oxygen levels Offer a clearer image with less background noise than found in typical microscopy

Figure 1 - The principle behind confocal microscopy, the preferred technique for fluorescence microscopy

Disadvantages;

Background emission via autofluorescence is possible due to the natural fluorescence of many large, highly conjugated biomolecules

Self-quenching

Photobleaching, the photochemical loss of agent molecules Biocompatibility and toxicity, this technique often requires heavy metals Transport-membrane permeability and active uptake can be difficult to achieve Tissue damage and penetration are inversely proportional, UV light offers the best

penetration but will also trigger cell death due

The photophysics of a fluorescence imaging agent are of utmost importance, of particularimportance are the Stokes shift and the luminescence lifetime .

The Stokes shift (Figure 2) is the difference, in wavelength, between the absorption and emission of a fluorescent molecule. The larger this shift, the easier it is to avoid autofluorescence (typically,


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autofluorescence has a Stokes shift of


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Figure 3 - The Jablonski diagram for a 3MLCT fluorescence process

Iridium Lumophores

Figure 4 - The structures of two Iridium lumophores

Tris-cyclometallated ppy complexes have a high luminescence intensity but are more difficult to tuneand so bis-cyclometallates, using ppy and N^N complexes, that allow the introduction of groups totune the fluorescence and importantly the biology are preferred. These systems are already cationicwhich is appropriate for transfer across cell membranes. Ir-bis-cyclometallates are synthesised from

iridium chloride (Figure 5) and go via a bridged bis-ppy bis-( 2)chloro complex, the benefit of thisapproach being that in the final step the R group can be chosen to tune the properties as desired. Bychanging the ppy unit (Figure 6) the photophysics can be tuned with emission possible from theentire range of the spectrum, the lifetime is proportional to the energy gap and ranges from 3000 400ns.

There are over 20 examples in the literature of iridium lumophores that show general cytoplasmicstaining with a limited number of these showing poorly controlled localisation in the nucleus. Thereare also two examples of analogous rhodium systems that both show cytoplasmic staining.

ISC

d

Singlet excited *

Stokes ShiftTriplet

pi

d-d transitionforbidden


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Figure 5 - The synthesis of Ir-bis-cyclometallates

max = 450nm max = 506nm

max = 597nm

Figure 6 - Example iridium lumophores and their emission wavelengths


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Ruthenium LumophoresRuthenium dyes often take the same form as iridium dyes, only the synthesis requires an additionalprecaution. The reaction is saturated with lithium chloride to prevent the formation of mixedproducts, in Figure 7 X is the tuneable moiety.

Figure 7 - Synthesis of a ruthenium lumophore

Rhenium LumophoresRhenium lumophores are built around a similar general design (Figure 8) , but with only one bipy orphenathroline ligand capable of MLCT, substituted pyridine is again used to tune the localisation anduptake of these lumophores providing rapid access to a library of agents. Where R is a largelipophilic moiety (e.g. a C 13 fatty acid), the imaging agent is likely to accumulate in membranes,

smaller lipophilic units (e.g. cyclohexyl) are capable of entering the nucleolus.

Figure 8 - The general structure of a rhenium lumophore

With only one large unit complexed to the rhenium metal the core is smaller than that of iridiumimaging agents.


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Figure 9 - Synthesis of a rhenium lumophore

The AgPF6 used in the synthesis of rhenium complexes (Figure 9) is weakly coordinating andprecipitates silver chloride, this is to increase the efficiency of the reaction, in turn limiting the wasteof any pyridine.

The reaction can be monitored via the emission as the electronegativity of the nitrogen, whichimpacts upon the energetics of the conjugated system (donation of electrons raises the energiesinvolved), changes upon coordination although this effect may be weak. This also has some uses invivo although these are limited.

A particularly important class of rhenium agents are those that target the mitochondria. Thisorganelle is protected by an internal membrane with a high potential barrier but being able to studyand image the mitochondria is vital as it can be central in many diseases. Mitochondria have a highthiol concentration, a fact that rhenium agents take advantage of MitoTracker (Figure 10) is acationic, lipophilic and thiol reactive molecule that, once inside the organelle, will form a fixed

adduct with glutathione, a polar membrane- impermeant, thus holding the imaging agent within themitochondria.

Figure 10 - MitoTracker Red and glutathione


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There is one report of a dimeric rhenium agent (Figure 11) that shows MLCT, conjugates withpeptide nucleic acids (PNAs) and shows nuclear accumulation as well as having an emissionwavelength sensitive to the environment.

Figure 11 - Dimeric rhenium imaging agent


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Figure 12 - A summary of the use of d -block lumophores as fluorescence imaging agents


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f -block LumophoresLanthanides behave as Lewis acids, they form a sphere of positive charge and, due to how diffusethe f -orbitals are, there is no overlap with the ligand lone pairs. They are therefore hard ions andprefer hard donors, the ligands are organised entirely due to stearic interactions and the bonds aremostly electrostatic and, consequently, labile. Stability is achieved in lanthanide chemistry throughthe use of large chelating systems.

The crystal field splitting in lanthanide ions is negligible. Unlike d -block elements where it isresponsible for the spectra we see, in f-block chemistry the emission is due to spin-couplinginteractions. An interesting result of this is emission spectra that show peak wavelengths dependentonly upon the metal ion in question, i.e. Eu 3+ will always have emissions at characteristicfrequencies. The intensity of the emission is affected by the nature of the solvent and ligand system,and different systems will provide different fine splitting and so, using these fingerprints, f -blocklumophores can provide useful information on the environment.

To optimise the intensity of the emitted light an f -block lumophore will usually contain an antennaemolecule (Figure 13) . Since the f-f transition is forbidden an organic antenna that first absorbsenergy before passing it to the metal triplet state greatly enhances the practicality of using an f-block lumophore. The antenna used may also be built on d -block (Figure 14) functionality asopposed to purely organic antenna in which the transfer of energy is d to f triplet-triplet exchange.The result is a lumophore with a large Stokes shift and a very long lifetime, however, designing anantenna can be complex as it must have a higher energy singlet and triplet state than the lanthanideand so care must be taken in designing these systems. Design is further complicated by biologicalconditions, in which the large amount of environmental Ca 2+ ions can lead to ion exchange resultingin toxicity from the lanthanide, this is a factor that is easy to overlook in the laboratory.

Figure 13 - Simple Jablonski diagram showing the action of an antennae in f -block lumophores

f -block agents are much more suitable than d -block counterparts for time-gated experiments as aresult of the long lifetime. They are also often near-infrared (NIR) emitters, ideal for use in vivo asNIR wavelengths easily penetrate the body while being at a low enough energy to not harm cells orinterfere with cellular processes. There is a slight trade-off common to photophysics, in like systemsthe lifetime of the fluorescence will decrease as the NIR character of the emission increases, evenfurther complicating ligand design.

An

1An

3An

Ln*

LnAntenna Lanthanide

energy transfer


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The lanthanide complexes used in imaging are often not cationic but neutral, instead of passivediffusion across the potential barrier at the cell membrane they are instead taken into the cells byactive transfer processes. The importance of active processes is proven by a simple experiment, byincubating the imaging agent with cells at 4 oC, a temperature at which active transfer processes willbe almost entirely stopped, and then raising the temperature back to 37 oC. If it can be shown thatthe agent enters the cell at body temperature, and if at 4 oC there is very little, if any, transfer theagent is being transported by active processes.


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Figure 14 - The synthesis of an f -block imaging agent that utilises a d -block antenna


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The Synthesis of f -block Imaging AgentsThe synthesis of these agents is often complicated and step-wise (Figure 15) , although thecoordination chemistry itself is simple, often involving little more than addition of the lanthanidehalide and heating the solution.

Figure 15 - Synthesis of an f -block agent

In order to guarantee a ligand system that will successfully retain the lanthanide ion and providesuitable photophysics while maintaining tuneable biological properties there is often a need toprotect and deprotect different moieties. The ligand design will often incorporate several

carboxylates or other hard donors to saturate the coordination at the metal; this is in order to


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prevent the complexation and subsequent vibration of water molecules that would otherwisequench fluorescent processes.

Frster Resonance Energy Transfer (FRET)If two species have overlapping absorption/emission spectra it is possible for non-radiative, distance

dependent (typically


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Figure 17 - A PET scanner

Table 2 - Isotopes common to PET imaging

Isotope life (hrs) Bio-relevant oxidation states; typical ligand families in biomedicalapplications

11 C 0.3 N/A18F 1.8 N/A64Cu 13 Cu +, Cu2+, ATSM, polymine, polycarboxylate99m Tc 6 Tc+, TcV; tren, dipicolylamine186/188 Re 89, 17 Re +, ReV; tren, dipicolylamine, CO (PET); polypyridyls, CO (confocal

microscopy)111 In 67 In 3+ polymine, polycarboxylate86Y 15 Y3+ polymine, polycarboxylate66/67/68 Ga 10, 78, 1 Ga 3+ polymine, polycarboxylate201 Tl 73 Tl3+ polymine, polycarboxylate89Zr 78 Zr4+; polyanion, polyamine polyol (PET); cyclopentadienyl derivatives

(cytology)117

Lu 160 Lu3+

polymine, polycarboxylate


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64Cu PET Agents

64Cu is a positron emiutter with a half-life, t 1/2 , of 13 hours. Since Cu2+ is d 9 and therefore Jahn Teller

active, most agents are designed to be square planar with the two axial sites left free for water tocoordinate (the rate of ligand exchange at the axial sites is practically diffusion controlled),

otherwise macrocycles can be used to contain the metal. Copper is best suited to medium donorligands, e.g. sulphur and nitrogen in the form of imines, Schiff bases and pyridines, and many arebased on Cu-ATSM (Figure 18) . Derivatives of Cu-ATSM are able to localise in hypoxic cells such astumour cells, which grow so fast they are unable to maintain the required blood flow. It is suggestedthat in hypoxic cells the Cu 2+ is reduced and unlike in healthy cells there is little re-oxidation, thecationic Cu + species is then retained in the cells. Protonation may also explain how the localisationoccurs as tumour cells are usually acidic.

Figure 18 A Cu-ATSM derivative

These species are synthesised by transmetallation of luminescent zinc analogues, this is a rapidprocess ensuring little loss of radiochemical yield (RCY). The luminescence of the zinc complexes isdue to the organic fluorophore, the zinc is only responsible for holding the fluorophore on a plane.Copper is even more effective at holding the surrounding ligand in a plane, however d 9 paramagneticcopper is far too effective at quenching fluorescence. The zinc analogues have the potential toperform bimodal imaging in conjunction with the copper complexes.

Single Photon Emission Computed TomographySPECT imaging is similar to PET except it employs agents that decay by -emission.

99m Tc SPECT Agents

There are two main families of 99m Tc agent;

TcI(CO)3 units TcV complexes, usually L 4Tc=O where L= nitrogen, oxygen or sulphur

Technetium is often generated as pertechnetate, Tc VII, which is then reduced to a biocompatibleform in the presence of a ligand to form a membrane permeable complex.

TcI complexes are often derived from bispicolylamine which can be incorporated into bioactive unitsusing peptide coupling through the single amino acid chelate (SAAC), upon complexation with

TcI(CO)3(aq) 3 (generated from TcVIIin situ via TcO 4

- (aq) + HC(O)BH2 [Tc(CO)3(H2O)3]I) the active agent

is formed (Figure 19) .


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Figure 19 - Synthesis of Tc I(CO)3L3 agents

Bimodal ImagingBimodal imaging is the use of a single probe that utilises two different imaging modalities. Thiswould allow for one agent that can image both the whole body and at a cellular level without thefear of false results, with separate agents the localisation may not be congruent.

Using combined radio and fluorescence imaging agents is perhaps the ideal bimodal approach sinceradio imaging works on the whole body but with a low resolution, perfectly complimenting the highresolution provided by fluorescence. This could be used to guide surgeons or to diagnose morespecific biochemistry within a tumour. Both radio and fluorescence depend on photon detection andso potentially both could be detected simultaneously.Several issues arise, the ligands for the radioisotope and fluorophore must be robustly linked, and itmay be difficult to define a doseage since both techniques rely on different concentrations ingeneral.

One example of a potential bimodal agent is a Cu-ATSM-pyrene complex (Figure 20) that, unusually,does not show fluorescence quenching by the d 9 Cu2+.

Figure 20 - A Cu-ATSM-pyrene bimodal imaging agent

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CH2402 - Imaging Agent Design